The long-term goal of our labs is to determine the molecular mechanisms by which post-translational modifications of histones regulate gene expression and DNA repair. These two cellular processes are vital for the growth and health of all organisms. Alterations in post-translational histone modifications affect the regulation of gene expression and DNA repair and can lead to diseases such as ICF, Rett and ATRX syndromes and, most predominantly, cancer. This research project will determine the molecular mechanisms by which four key post-translational modifications at histone H3 residues K56, K115, T118 and K122 function. These modifications were recently identified in structured regions of the nucleosome. They occur both individually and together in vivo, and are critical for transcriptional regulation and DNA repair. The mechanisms by which these modifications carry out vital biological functions remain poorly understood. However, each modification is buried beneath DNA in the histone-DNA interface at one of two critical regions of the nucleosome: the dyad symmetry axis and the DNA entry-exit region. This implies that the mechanisms by which these modifications function must require significant changes in nucleosome conformation and/or dynamics. Therefore, combined biochemical and biophysical studies are key to understanding the role of these modifications. The four modifications will be studied by reconstituting uniformly modified semi-synthetic nucleosomes using histone proteins that are constructed by expressed protein ligation and by sequential chemical ligation. Multiple approaches will be used to quantify modification-induced changes in chromatin conformation and dynamics: fluorescence resonance energy transfer, restriction enzyme digestions, nucleosome mapping, nucleosome competitive reconstitutions, stopped flow fluorometry and fluorescence correlation spectroscopy. These experiments will determine whether and how these four histone H3 modifications in the DNA-histone interface regulate, in Aim 1, Nucleosome Structure and DNA site Exposure, in Aim 2, Nucleosome Positioning and Stability and, in Aim 3, Nucleosome Dynamics. The successful completion of this research project will make a major impact on scientific knowledge and the molecular understanding of disease in two ways. First, it will determine how four modifications in histone H3 buried within the nucleosome DNA-histone interface facilitate both DNA repair and transcriptional regulation. Second, it will provide insight into the possible mechanisms of over 20 post-translational histone modifications that are known to be buried throughout the nucleosome DNA-histone interface.